8 research outputs found
A Computational and Experimental Study of the Regulatory Mechanisms of the Complement System
The complement system is key to innate immunity and its activation is necessary for the clearance of bacteria and apoptotic cells. However, insufficient or excessive complement activation will lead to immune-related diseases. It is so far unknown how the complement activity is up- or down- regulated and what the associated pathophysiological mechanisms are. To quantitatively understand the modulatory mechanisms of the complement system, we built a computational model involving the enhancement and suppression mechanisms that regulate complement activity. Our model consists of a large system of Ordinary Differential Equations (ODEs) accompanied by a dynamic Bayesian network as a probabilistic approximation of the ODE dynamics. Applying Bayesian inference techniques, this approximation was used to perform parameter estimation and sensitivity analysis. Our combined computational and experimental study showed that the antimicrobial response is sensitive to changes in pH and calcium levels, which determines the strength of the crosstalk between CRP and L-ficolin. Our study also revealed differential regulatory effects of C4BP. While C4BP delays but does not decrease the classical complement activation, it attenuates but does not significantly delay the lectin pathway activation. We also found that the major inhibitory role of C4BP is to facilitate the decay of C3 convertase. In summary, the present work elucidates the regulatory mechanisms of the complement system and demonstrates how the bio-pathway machinery maintains the balance between activation and inhibition. The insights we have gained could contribute to the development of therapies targeting the complement system.Singapore. Ministry of Education (Grant T208B3109)Singapore. Agency for Science, Technology and Research (BMRC 08/1/21/19/574)Singapore-MIT Alliance (Computational and Systems Biology Flagship Project)Swedish Research Counci
Local Inflammation Induces Complement Crosstalk Which Amplifies the Antimicrobial Response
By eliciting inflammatory responses, the human immunosurveillance system notably combats invading pathogens, during which acute phase proteins (CRP and cytokines) are elevated markedly. However, the Pseudomonas aeruginosa is a persistent opportunistic pathogen prevalent at the site of local inflammation, and its acquisition of multiple antibiotic-resistance factors poses grave challenges to patient healthcare management. Using blood samples from infected patients, we demonstrate that P. aeruginosa is effectively killed in the plasma under defined local infection-inflammation condition, where slight acidosis and reduced calcium levels (pH 6.5, 2 mM calcium) typically prevail. We showed that this powerful antimicrobial activity is provoked by crosstalk between two plasma proteins; CRP∶L-ficolin interaction led to communication between the complement classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition consistently proved the functional significance of the amplification pathways in boosting complement-mediated antimicrobial activity. The infection-inflammation condition induced a 100-fold increase in CRP∶L-ficolin interaction in a pH- and calcium-sensitive manner. We conclude that the infection-induced local inflammatory conditions trigger a strong interaction between CRP∶L-ficolin, eliciting complement-amplification pathways which are autonomous and which co-exist with and reinforce the classical and lectin pathways. Our findings provide new insights into the host immune response to P. aeruginosa infection under pathological conditions and the potential development of new therapeutic strategies against bacterial infection
Aggregation and fusion of lipid vesicles induced by diphtheria toxin at low pH: Possible involvement of the P site and the NAD+ binding site
Construction of a toxic insulin molecule: Selection and partial characterization of cells resistant to its killing effects
The effect of monensin on gametogenesis and zoosporogenesis in the aquatic fungus,Allomyces macrogynus
Association between baseline levels of C-reactive protein (CRP) and a dinucleotide repeat polymorphism in the intron of the CRP gene
Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis
Human C-reactive protein (CRP), as a mediator
of innate immunity, removed damaged cells by activating
the classical complement pathway. Previous studies have
successfully demonstrated that CRPs are differentially induced
as glycosylated molecular variants in certain pathological
conditions. Affinity-purified CRPs from two most
prevalent diseases in India viz. tuberculosis (TB) and
visceral leishmaniasis (VL) have differential glycosylation
in their sugar composition and linkages. As anemia is a
common manifestation in TB and VL, we assessed the
contributory role of glycosylated CRPs to influence hemolysis
via CRP-complement-pathway as compared to
healthy control subjects. Accordingly, the specific binding
of glycosylated CRPs with erythrocytes was established by
flow-cytometry and ELISA. Significantly, deglycosylated
CRPs showed a 7–8-fold reduced binding with erythrocytes
confirming the role of glycosylated moieties. Scatchard
analysis revealed striking differences in the apparent binding constants (104–105M−1) and number of binding
sites (106–107sites/erythrocyte) for CRP on patients’ erythrocytes
as compared to normal. Western blotting along with
immunoprecipitation analysis revealed the presence of
distinct molecular determinants on TB and VL erythrocytes
specific to disease-associated CRP. Increased fragility, hydrophobicity
and decreased rigidity of diseased-erythrocytes
upon binding with glycosylated CRP suggested membrane
damage. Finally, the erythrocyte-CRP binding was shown to
activate the CRP-complement-cascade causing hemolysis,
even at physiological concentration of CRP (10μg/ml).
Thus, it may be postulated that CRP have a protective role
towards the clearance of damaged-erythrocytes in these two
disease